Targeted hydrophilic polymer, binders with interferon and medical composite comprising above binders

ABSTRACT

The present invention relates to an active targeted water-solubility macromolecule polymer, conjugate With interferon and pharmaceutical composition comprising the conjugate. The targeted agent includes, for example, glucose, galatose and the like, as well as their derivates. The conjugate of the present invention is well in water-solubility and havc long physiological cycle half-life period, and have specific recognition to pathology organize, improved and increased medication effect of interferon to Hepatitis B, Hepatitis C etc. infectivity sickness and cancer, infect complication etc.

FIELD OF THE INVENTION

The present invention relates to a targeted hydrophilic polymer,conjugate with interferon, and composition comprising the conjugate. Thetargcting agent in the invention is saccharide derivatives, such asamino-glucose, amino-galactose and the like.

BACKGROUND OF THE INVENTION

Interferons are a group of natural proteins that occur in the celldivision process and exhibit anti-viral, antitumor, and immunomodulatoryactivities. The recombinant human interferon α and interferon β havebeen used as drugs for treatment of viral infections, cancer andmultiple sclerosis. In the treatment of both hepatitis C and hepatitisB, interferon ox is the only effective agent on the market, and hasbecome the standard therapy. However, because of its short circulationhalf-life (4-8 hours), their anti-viral activity and long-term treatmenteffect are limited. Recently, Schering-Plough and Hoffmann-La Roche haveindependently developed long-lasting interferon a formulations byconjugating with a synthetic water-soluble polymer. In 2001,Schering'sproduct, PEG-intron®, obtained approval from the US FDA for treatment ofhepatitis C. Roche's product, Pegasys®, had finished clinical studiesand is waiting for the US FDA's approval In WO01/74399A1, a targetedsystem using galactose-PEG was disclosed. Although it can obtain theeffect of targeting toward the suffered tissue for interferon, but themeasures for applying it are very complicated and difficult to meet theindustrial needs. In addition, the polymer cannot be conjugated withinterferon in an aqueous phase, and the conjugation must be catalyzedwith enzyme.

Polyethylene glycol has been widely used in the field of proteinconjugation. The resulting conjugates will improve in vivo circulationhalf-lives, and reduce immunogenecity and toxicity for proteins,peptides and other therapeutics. In both PEG-intron® and Pegasys®,polyethylene glycol not only improves their anti-viral efficacy but alsoreduces their injection frequency from three times a week to once aweek.

However, neither PEG-intron® nor Pegasys® can achieve greater than 60%patient sustained response rate. Especially for hepatitis C virus ofgenotype I, sustained response rate is lower than 42%. To furtherimprove the efficacy of interferon, it is believed that targeteddelivering more interferon to the virus infected tissues or organs couldbe very beneficial.

In the researches of glycobiology, many bio-macromolecules on the cellsurface are glycosylated compounds, also referred to glycoconjugate.They can be further classified into three types: glycoproteins,protcoglycans and glycolipids. Glycosylated compounds with extendingoligosaccharides are the typical components anchored on the surface ofcell membrane, cell wall and organella. The extending sugar parts havefunctions of information coding, which play very important roles incell-cell recognition. For example, the recognition between a sperm andan egg in reproduction process is modulated by the sugar-proteininteractions.

Polysaccharides are not the direct products of gene transcription, buttheir biosynthesis is adjusted by post translation. When there is adefect in the cell adjusting mechanism (such as in auto-immune diseases,cancer or other gene-related diseases), sugar synthesis and degradationare altered, resulting in changes in the component and composition ofthe sugar on the cell surface, leading to the alternation of the wholeimmune system. Comparing to amino acids, nucleotides, and fatty acids,the structures of saccharides are more complicated. For example, twodifferent amino acids can produce two kinds of dipeptides, while twomonosaccharides can produce eleven kinds of disaccharides. Three aminoacids can produce six kinds of tripeptides, while three monosaccharidescan lead to 1,056 kinds of trisaccharides. It is due to the branchingproperties of saccharide structure, which differs polysaccharides fromothcr bio-macromolecules (such as proteins and polynucleotides). Basedon its inherent structural complication, polysaccharides have become thecarriers of detailed biological information, playing an important rolein cell recognition and signal transduction.

Liver cancer is one of the common malignant cancer. Very few patientscan be treated with surgical removal, and at same time, chemical andradical therapies have very low response rate. In China, liver cancerresults in 100,000 deaths every year, and becomes the leading deathcause in some parts of China. The major aim of the present invention isto improve the efficacy and lasting effect of the medicines toward liverdiseases.

The asialoglycoprotein receptor (ASGPR) is a trans-membrane glycoproteinonly existing on the liver cells of mammals. ASGPR can specificallyrecognize and then binds with the glycoproteins having a galactose unitat the terminal. The formed ligand-receptor complex can then beinternalized into lysosomc, and the macromolecule can be releasea ASGPRitself will not be degraded, but recycle back to the surface of the cellmembrane to participate into next cycle of transportation. Because ofits specific property, ASGPR is also called liver specific galactose(H-Gal) receptor.

It is believed that with the combination of targeting agent andhydrophilic polymer, the interferon level at the infected tissues andorgans will be increased, which will improve the anti-virus effect andachieve an improved treatment efficacy. In the treatment of hepatitis Cand B, the efficacy of interferon will be significantly increased byextending its circulation half-life with PEGs and redirecting itsdistribution in the body, especially in the liver by the targetingagents. As a result, it is expected that anti-viral efficacy fortreatment of hepatitis C and hepatitis B will be increased.

SUMMARY OF THE INENTION

Therefore, one objective of the present invention is to provide anactivated targeted hydrophilic high-molecular weight polymer derivativesrepresented by the following formula, which can react with protein (e.g.interferon) in an aqueous phase without enzymes or other catalysts:

wherein:

-   X and Y are linking groups, selected from NH or O;-   P is a hydrophilic polymer having an average molecular weight more    than 18 kDa;-   T is a targeting agent of a saccharide or its derivatives, such as    glucose, galactose and the like;-   F is an activated group, which can react with amino or thiol groups    of the proteins in an aqueous phase without enzymes or other    catalysts.

In a preferred embodiment of the invention, the activated targetedhydrophilic high-molecular weight polymer derivatives are represented asfollows:

wherein:

-   X is a linking group selected from NH or O;-   P is a bydrophilic polymer having an average molecular weight more    than 18 kDa;-   T is a targeting agent of a saccharide or its derivatives, such as    glucose, galactose and the like.

In another aspect of the invention, there is provided a conjugate of thetargeted hydrophilic polymer and an interferon molecule represented asfollows:

wherein:

-   PEG is a polyethylene glycol having an average molecular weight more    than 18 kDa;-   T is a targeted group of a saccharine or its derivatives, such as    glucose, galactose and the like;-   Interferon is a protein of interferon family.

In the invention, the interferon can be either of α-, β- orγ-interferon.

In the invention, the tareting agent is of a saccharide or itsderivatives, including monosaccharide such as glucose, mnaose,galactose, lactose and fructose, and their oligomers.

In the invention, the hydrophilic polymer is selected from apolyethylene glycol, a polypropylene glycol, a polyvinyl alcohol, apolyacrylmorpholine and the copolymers thereof. Among them, apolyethylene glycol is preferred.

In the invention, the activated group F is selected from the groupconsisting of an ester group, a carbonate group, an amide group, anamido ester group, an ether group, a cabamate group, an acetal group andthe like.

In one aspect of the invention, there is provided a pharmaceuticalcomposition comprising the conjugate according to the invention. Thecomposition can be used for treatment of infectious diseases such ashepatitis C and hepatitis B, cancer and infective complications throughsubcutaneous or intravenous injection.

The conjugate according to the present invention includes a highmolecular weight hydrophilic polymer and at least one targeting agent.The high molecular weight hydrophilic polymer will provide an extendedcirculation half-life, while the targeting agent will lead interferon tothe disease tissues. In the treatment of hepatitis C and hepatitis B,the targeting agents can be saccharide derivatives or antibodiestargeting toward liver. Therefore, in the present invention, theconjugate of the polymer and interferon will not only provide prolongedcirculation half-life, but also improve the interferon concentrationaround the infected liver tissues. It could significantly improve thetreatment efficacy for hepatitis C than the current PEG-interferonconjugates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention utilizes galactose property which can bespecifically recognized and binded by liver surface asialoglycoproteinreceptor (ASGPR), to further develop liver targeted drug deliverysystem.

In the present invention, the targeted conjugate can be prepared asfollows: the hydrophilic polymer is modified to introduce a targetedagent and a activated functional group into different terminals of thepolymer, and then the activated polymer is conjugated with aninterferon, such as α-, β- or γ-interferon. Hereafter, PEG is used as anexample of the hydrophilic polymer to illustrate the present invention.It should be understood that the hydrophilic polymer in the conjugate ofthe present invention can not be limited to a polyethylene glycol andits copolymer, it also can be a polypropylene glycol, a polyvnylalcohol, a polyacrylmorpholine or the copolymers thereof.

The general structure of PEGs is shown as the formula below:

wherein:

-   R is H or a C₁₋₁₂ alkyl group; and-   n is an integer, representing the degree of the polymerization.

In respect of PEGs, they are usually measured by molecular weight. It ispreferred that the molecular weight of PEG which forms the conjugatesfalls in the range from 300 to 60000 Daltons, which means n is about 6to 1300. It is more preferred that n is 28, 112 and 450, respectivelycorresponding to molecular weight of 1325, 5000, and 20000. Because ofthe potential non-homogeneity of the starting PEGs which are usuallydefined by their molecular weights rather than the self-repeating unitn, PEGs are normally characterized with a weight average molecularweight, rather than their self-repeating units represented by n. Thestarting PEG compounds with different molecular weights are readilysynthesized using inethods known in the art or they are commerciallyavailable.

In addition of a linear polymer, polymers having a branched or otherstructure, such as Y shape, U shape and the like, can also be used forthe modification of an interferon molecule. The structure of the polymermay be chosen depending on the properties of the pharmaceuticalmolecules.

In the present invention, the targeted hydrophilic polymer is based on apolyethylene glycol as a staking material, and is synthesized by bindinga targeted agent, such as glucose, mannose, galactose, lactose andfructose, to the PEG according to any synthetic method well known to theskilled person in the art. In the practice, one terminal group of a PEGis activated first, and then reacts with an amino group or a hydroxylgroup of a saccharide molecule. At the other end of a PEG, activationwill be done to ensure that the targeted polymer system can react withdrug molecules to form conjugates.

A carboxyl group is a conunon fimctional group for the PEG activation.There are many ways to incorporate a carboxyl group into a PEG Forexample, an OCH₂COOH structure at the terminal of PEG can be obtained asfollows. This functional group can form an active ester that can easilyreacts with an amino group or a hydroxyl group present in a sugar unit.

Also, an OCOOH functional group can be obtained as follows:

In the process of conjugating a saccharide molecule, N-hydroxylsuccinimide (NHS) can be used to activate the carboxylic group. Theester of N-hydroxyl succinimide has a high reactivity toward an aminogroup to form a corresponding amide.

After reaction, it is necessary to isolate and purify the product. Ananion exchange resin may be used to obtain the desired compound.

The conjugates of the present invention can be administered in the formof pure compounds or suitable pharmaceutical compositions, via anyacceptable routes or being included in a reagent for similar use. Thus,the conjugates can be administered via oral, nasal, parenteral, topical,transdermal, rectal or injection routes in the form of solid, semisolid,lyophilized powder or liquid, for example, tablets, suppositories,pills, soft and hard gelatin capsules, powder, solution, suspention andaerosols. Preferably the unit dosage form is suitable for aprecise-dosage and easy administration. The composition includesconventional pharmaceutical carriers or excipients and the conjugate(s)of the present invention as the active ingredient(s). Furthermore, italso can include other agents, carriers and excipients.

Generally speaking, depending on the method of administration, thepharmaceutically acceptable compositions will include about 1-99 wt. %of the conjugate of the present invention, and 99-1 wt. % of anysuitable pharmaceutical excipient; Preferably they include 5-75 wt. % ofthe conjugate and the rest is any suitable pharmaceutical excipient.

The preferable way of administration is injection with a general dailydosage scheme, wbhich can be adjusted based on the severity of thedisease to be treated. The conjugates of the present invention or theirpharmaceutically acceptable salts may be formulated in the dosage forinjection by, for example, dissolving 0.5-50% of the active componentsin a liquid pharmaceutical carrier, such as water, saline, aqueousglucose, glycerol, ethanol and the like to form a solution orsuspension.

The compositions which can be administered as liquid such as solutionsand suspension can be prepared by dissolving and dispersing theconjugate of the present invention (about 0.5-20%) and optionally apharmaceutical excipient into a carrier. Example of carriers includeswater, saline, aqueous glucose, glycrol, ethanol and the like.

If needed, the phariaceutical composition of the present invention canfurther include an adjuvant in a small amount, such as a wetting agent,an emulsifier, a pH buffer, an antioxidant and the like. For example,citric acid, sorbitan monolaurate, triethanolaminc oleate, butylatedhydroxytoluene and the like can be added.

The practical preparation methods of such dosage forms are known orobvious to the skilled in the art. For example, see Ramington'sPharmaceutical Sciences, 18_(th) edition, (Mack Publishing Company,Easton, Pa., 1990). In any case, according to the techniques of thepresent invention, the composition applied will include an effectiveamount of the conjugate of the present invention for the treatment ofcorresponding disease.

EXAMPLES

The polymer derivatives and the conjugates of the present invention andtheir preparation methods will be further described by the followingexamples. These examples do not intend to limit the scope of theinvention by any means. The scope of the present invention can bedetermined by the claims.

Example 1 Preparation of Polyethylene Glycol Diethyl Acid

In an azeotropic distillation device, poly(ethylene glycol) of Mw 20000(PEG20000, 100 g, 5 mmole) was dissolved in 500 ml of benzene at 50° C.and refluxed under N₂. After two hours, tbe solution was distilled toabout 300 ml and was then cooled to 35° C. To the solution was addedpotassium tert-butoxide (50 mmole) dissolved in a mixed solvent oftert-butanol (100 ml) and benzene (50 ml). The mixture was stirred fortwo hours. To the mixture was added tert-butyl bromoacetate (60 mmole).The resulting solution was stirred under N₂ at room temperatureovernight. The formed salt was then removed by filtration, the filtratewas concentrated by rotary evaporation, and the residue was then addedinto 500 ml of isopropyl alcohol (IPA). The precipitate was collected byfiltration and dried under vacuum. The dried solid was dissolved into abasic solution of pH 12.0, and the solution was stirred overnight tocomplete hydrolysis. The pH of the solution was adjusted to 2.5 byaddition of 1 N aqueous HCl solution, and the solution was extractedthree times with 200 ml of methylene chloride. The combined organicphases were dried over anhydrous sodium sulfate, filtered, concentratedon a rotary evaporator, and then poured into 500 ml of IPA. The productwas collected by filtration and dried under vacuum. Yield 90 g (90%).NMR (DMSO): δ3.5 (H in PEG, br m), 4.00(2H, s).

Example 2 Preparation of Glucosamine-Polyethylene Glycol Ethyl Acid

PEG20000-bis-acid (80 g, prepared in Example 1) was dissolved in 500 mlof anhydrous methylene chloride. To the solution was added 1.0 g ofn-hydroxylsuccinimide (NHS) and 2.0 g of dicyclohexylcarbodiimide (DCC).The solution was stirred at room temperature overnight. The solution wasfiltered and the solvent was removed by rotary evaporation. The residuewas added to 300 ml of isopropyl alcohol (IPA). The precipitate wascollected by filtration, washed with 20 ml of dietbyl ether and driedunder vacuum. Yield 72 g (90%). NMR(DMSO): δ3.5 (H in PEG, br mn), 4.60(2H, s), 2.81 (4H, s).

The PEG20000-bis-acid NHS ester (70 g) was dissolved in 200 ml ofanhydrous methanol. To the solution was added 1 g of glucosamine and 1ml of dried triethylamine (TEA). The solution was stirred at 35° C. overnight. The solvent was removed under vacuum at temperature below 35 ° C.To the residue was added 250 ml of sodium carbonate solution (5 wt %).The solution was stirred overnight. The pH of the solution was adjustedto 3 with HCI solution (1 N), and the solution was extracted three timeswith 300 ml of methylene chloride. The combined organic phases weredried over anhydrous sodium sulfate, filtered, concentrated on a rotaryevaporator, and then precipitated into 500 ml of IPA. The product wascollected by filtration and dried under vacuum. Yield: 61 g (87%).

The mixture of glucosamine-PEG20000-acids (30 g) was dissolved indeionized water and separated via an anion-exchange column. The fractionof glucosamine-PEG20000-monoacid was collected. Yield: 15 g (50%).Melting point: 60-64° C.

Example 3 Preparation of N-Hydroxyl Succinimide Glucosamino-PolyethyleneGlycol-Mono Acid Ester (I)

Glucosamine-PEG20000-mono-acid (from Example 2) (8 g) was dissolved in80 ml of anhydrous methylene chloride. To the solution was added 50 mgof N-hydroxylsuccinimide (NFS) and 95 mg of dicyclohexylcarbodiimide(DCC). The solution was stirred at room temperature overnight. Thesolution was filtered and the filtate was concentrated by rotaryevaporation. The residue was added to 150 ml of isopropyl alcohol (IPA).The precipitate was collected by filtration, washed with ethyl acetateand dried under vacuum. Yield 7.5 g (94%). NMR (DMSO): δ3.5 (H in PEQ brm), 4.60 (2H, s). 2.81 (4H, s).

Example 4 Preparation of N-Hydroxyl SuccinimideGalactosamino-Polyethylene Glycol-Mono Acid Ester (II) acid ester (11)

The procedures as described in Examples 2 and 3 were repeated with anexception that galactosamine was used instead of glucosamine.

Example 5 Preparation of N-Hydroxyl SuccinimideGalactosanino-Polyethylene Glycol-Mono Acid Ester and α-Interferon

N-hydroxyl succinimide galactosamino-polyethylene glycol-mono acid ester(II, molecular weight 20,000) was dissolved in 5 ml of bufferedα-interferon solution with interferon concentration of 5 mg/ml (pH 7.4).In the reaction solution, N-hydroxyl succinimidegalactosamino-polyethylene glycol-mono acid ester and α-interferon weremixed at a ratio of 5:1. The solution was shaken. for 3 hour at roomtemperature. The pH of the solution was adjusted to 4.5 and the solutionwas diluted to an α-interferon concentration of 0.5 mg/ml, and purifiedby cation-exchange chromatography. The fraction containing the conjugateof mono-substituted galactosamino-polyethylene glycol-α-interferon wascollected. SDS-PAGE showed that the product contained no freeα-interferon.

Example 6

This example is to illustrate the preparation process of a typicalpharmaceutical composition administered parenterally. The compositioncomprises the conjugate prepared in Example 5. Composition The conjugateof α-interfeon 50 mg 0.9% saline to 10 ml

The conjugate of α-interferon (the conjugate of mono-substitutedgalactosamino-polyethylene glycol-α-interferon) was dissolved in 0.9%saline to obtain 10 ml solution for subcutaneous or intravenousinjection, which was filtered through 0.2 μm membrane and packedaseptically.

1. An activated targeted hydrophilic high-molecular weight polymerderivative having the following formula,

wherein: X and Y are linking groups selected from NH and O; P is ahydrophilic polymer having an average molecular weight more than 18 Kda;T is a targeting agent group of a saccharide or its derivatives; and Fis an activated group, which can react with amino or thiol groups of theproteins in an aqueous phase without enzymes or other catalysts.
 2. Anactivated targeted hydrophilic high-molecular weight polymer derivativehaving the following formula:

wherein: X and Y are linking groups selected from NH or O; P is ahydrophilic polymer having an average molecular weight more than 18Kda;and T is a targeted agent of a saccharide or its derivatives.
 3. Thehydrophilic high-molecular weight polymer of claim 1, wherein thehydrophilic polymer P is selected from the group consisting of apolyethylene glycol, a polypropylene glycol, a polyvinyl alcohol, apolyacrylmorpholine and the copolymers thereof.
 4. The hydrophilichigh-molecular weight polymer of claim 3, wherein the polyethyleneglycol has an average molecular weight of 18,000 to 50,000.
 5. Thehydrophilic high-molecular weight polymer of claim 1, wherein thetargeting agent T comprises glucose, mannose, galactose, lactose,fructose, and their oligomers and derivatives.
 6. The hydrophilichigh-molecular weight polymer of claim 1, wherein the activated functiongroup F is selected from the group consisting of an ester group, acarbonate group, an amide group, an amido ester group, an ether group, acabamate group, and an acetal group.
 7. A conjugate of a targetedhydrophilic polymer and interferon having the following formula:

wherein: T is a targeted agent; P is a hydrophilic polymer having anaverage molecular weight more than 18 kDa; Interferon is a protein ofinterferon family.
 8. The conjugate of claim 7, wherein the hydrophilicpolymer P is selected from the group consisting of a polyethyleneglycol, a polypropylene glycol, a polyvinyl alcohol, apolyacrylmorpholine and the copolymers thereof.
 9. The conjugate ofclaim 8, wherein the polyethylene glycol has an average molecular weightof from 18,000 to 50,000.
 10. The conjugate of claim 7, wherein thetargeting agent T comprises glucose, mannose, galactose, lactose,fructose, and their oligomers and derivatives.
 11. The conjugate ofclaim 7, wherein the interferon is α-, β- or γ-interferon.
 12. Apharmaceutical composition comprising a conjugate according to claim 7and a pharmaceutically acceptable carder or excipient.
 13. Thecomposition of claim 12, wherein it may be formulated into the form forinjection or of solution, tablet, suspension and aerosol.
 14. Thecomposition of claim 13 for the treatment of hepatitis C and hepatitisB, cancer and infective complications.
 15. The hydrophilichigh-molecular weight polymer of claim 2, wherein the hydrophilicpolymer P is selected from the group consisting of a polyethyleneglycol, a polypropylene glycol, a polyvinyl alcohol, apolyacrylmorpholine and the copolymers thereof.
 16. The hydrophilichigh-molecular weight polymer of claim 2, wherein the targeting agent Tcomprises glucose, mannose, galactose, lactose, fructose, and theiroligomers and derivatives.
 17. The composition of claim 12, wherein thehydrophilic polymer P is selected from the group consisting of apolyethylene glycol, a polypropylene glycol, a polyvinyl alcohol, apolyacrylmorpholine and the copolymers thereof.
 18. The composition ofclaim 17, wherein the polyethylene glycol has an average molecularweight of from 18,000 to 50,000.
 19. The composition of claim 12,wherein said, the targeting agent T comprises glucose, mannose,galactose, lactose, fructose, and their oligomers and derivatives. 20.The composition of claim 12, wherein the interferon is α-, β- orγ-interferon.